Abstract Steroid hormones are a group of lipophilic hormones that regulate diverse physiological and pathological processes including immune response, energy homeostasis, sexual maturation and cancer progression. In contrast to the extensive literature describing their biosynthesis and signal transduction pathways, almost nothing is known regarding the mechanisms that regulate transport of steroids across the plasma membrane. This is due to the long-standing paradigm that lipophilic steroid hormones enter cells by simple diffusion across lipid bilayers. However, despite this dominant assumption, a simple diffusion model of steroid hormone trafficking is not supported by any conclusive in vivo evidence in any organism. The overall objective of this project is to demonstrate the function and significance of cell membrane transporters in steroid hormone trafficking and signaling in vivo, and to thereby challenge the conventional paradigm in endocrinology that steroid hormones freely travel across cell membranes by simple diffusion. The project will also screen chemicals that can inhibit steroid hormone entry into cells, with the goal of developing novel class of drugs and pest control reagents. The approach will first leverage the simple but powerful fruit fly model system to thoroughly investigate the functions of a putative importer for the steroid hormone 20-hydroxyecdysone (20E) that was discovered in preliminary studies. Functions of putative 20E exporters will also be analyzed, aiming to fully elucidate the molecular basis of transmembrane steroid hormone transport in vivo. The molecular basis and functional significance of transcellular steroid hormone transport will also be studied, using the blood-brain barrier of the fly central nervous system as a model. In the second step of the project, the yellow fever mosquito will be used to identify and characterize mosquito-specific 20E importers involved in development and reproduction. Lastly, chemical screening will be conducted to identify compounds that can block 20E entry into mosquito tissues, and their effects on mosquito development and reproduction will be tested in vivo. The project will apply diverse molecular genetic tools, leverage next-generation sequencing, and employ chemical screening to fully exploit the significance of our hypothesis. The steroid hormone importer analyzed in this project belongs to a family of solute-carrier transporters that exists in a wide variety of animals including humans, raising the possibility that transporter-mediated steroid hormone uptake is highly conserved among metazoans. Successful completion of this project may thus lead to novel clinical tools and pest control reagents, such as chemicals that can block the function of steroid hormone importers in a species- and tissue- specific manner.